Transcription factors represent a group of molecules within the cell that function to connect the pathways from extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes. One family of transcription factors, E2F transcription factors, regulates the expression of an extensive panel of genes that control DNA synthesis and cellular proliferation in a cell cycle-dependent manner reviewed in (Dyson, Genes Dev., 1998, 12, 2245-2262; Lavia and Jansen-Durr, BioEssays, 1999, 21, 221-230; Yamasaki, Results Probl. Cell. Differ., 1998, 22, 199-227).
The "E2F" designation given to these proteins refers to a multigene family of transcription factors that heterodimerize with members of the DP gene family (Helin and Harlow, J. Virol., 1994, 68, 5027-5035; Magae et al., J. Cell. Sci., 1996, 109, 1717-1726). E2F/DP heterodimers can mediate both transcriptional activation and repression and, to date, six members of the E2F family and two members of the DP family have been isolated.
The E2F family has been further subdivided into three subfamilies based on their interaction with the product of the retinoblastoma tumor suppressor gene (RB), a transcriptional repressor whose deregulation has been shown to result in oncogenesis. Consequently, the deregulation of the expression or function of E2F proteins alone or in conjunction with DP proteins has been implicated in the development of certain pathologic conditions, including cancer (Sladek, Cell. Prolif., 1997, 30, 97-105; Yamasaki, Biochim. Biophys. Acta, 1999, 1423, M9-15).
The first subfamily of E2Fs consists of E2F transcription factor 1, 2 and 3 (E2F-1, E2F-2 and E2F-3) which bind with high affinity to the retinoblastoma protein, pRB (Arroyo and Raychaudhuri, Nucleic Acids Res., 1992, 20, 5947-5954; Nevins, Science, 1992, 258, 424-429). The second subfamily comprises E2F transcription factors 4 and 5 (E2F-4 and E2F-5) and these bind to the pRB homologues, p107 and p130 while the third subfamily contains one member, E2F transcription factor 6 (E2F-6) that does not bind to any of the pRB family members.
While relatively little is known about the specific properties of the individual members of the E2F family, the best characterized are those of the first subfamily (E2F-1, E2F-2 and E2F-3). Because E2F binding sites are found in the promoters of many genes, these transcription factors mediate a multitude of cellular processes including apoptosis, cell cycle control, DNA replication, and proliferation (Bernards, Biochim. Biophys. Acta, 1997, 1333, M33-40; Helin, Curr. Opin. Genet. Dev., 1998, 8, 28-35; Nevins, Cell. Growth Differ., 1998, 9, 585-593).
It has been shown that when expressed alone in rat fibroblasts any of the three members of this subfamily can induce S-phase entry in serum starved cells (Lukas et al., Mol. Cell. Biol., 1996, 16, 1047-1057). In U343 astrocytoma cells, Dirks et al. have demonstrated that deregulated expression of any of the E2F transcription factors overcame cell cycle arrest and affects the expression of a distinct array of cell cycle regulatory proteins. However, it was further demonstrated that only the expression of E2F-1 or E2F-2 resulted in potent cell death (Dirks et al., Oncogene, 1998, 17, 867-876).
E2F transcription factors have been shown to regulate keratinocyte proliferation, implying a role in skin tumor development (Jones et al., J. Invest. Dermatol., 1997, 109, 187-193). In fact, it has been demonstrated that expression of five of the six members of the E2F family of transcription factors (excluding E2F-6) is upregulated in mouse skin tumors (Balasubramanian et al., Int. J. Oncol., 1999, 15, 387-390).
E2F transcription factor 3 (also known as E2F-3) is the third member of the E2F family identified from a low-stringency hybridization screen designed to isolate additional E2F-l-related genes (Lees et al., Mol. Cell. Biol., 1993, 13, 7813-7825). In these studies, Lees et al. have shown that the three E2F members all share four regions of protein sequence homology, each having a specific function. They also demonstrate that, while all three transcription factors are expressed in all cell lines examined, E2F-2 and E2F-3 are expressed at consistently lower levels than E2F-1. Upon examining human tissues, it was found that E2F-3 was expressed in all tissues except brain (Lees et al., Mol. Cell. Biol., 1993, 13, 7813-7825).
Despite similarities among E2F members, distinct functional roles have been identified in certain cell types. In studies of hematopoiesis, overexpression of E2F-1 in myeloid progenitor cells was shown to override survival signals in the cell provided by growth factors and lead to apoptosis. In contrast, overexpression of E2F-3 did not lead to apoptosis but to normal granulocyte differentiation (Strom et al., Cell Growth Differ., 1998, 9, 59-69).
The pharmacological modulation of E2F transcription factor 3 activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of E2F transcription factor 3 and investigative strategies aimed at modulating E2F transcription factor 3 function have only involved the use of antibodies. Consequently, there remains a long felt need for agents capable of effectively inhibiting E2F transcription factor 3 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of E2F transcription factor 3 expression.
The present invention provides compositions and methods for modulating E2F transcription factor 3 expression.